Compact biosensor of matrix metalloproteinase with cadmium free quantum dots

ABSTRACT

The invention provides a quantum dot (QD) modified optical fiber-based biosensor which characterizes matrix metalloproteinase (MMP) enzyme activity at pain signaling sites in the central nervous system (CNS) in vivo. Related systems and peptide biomarker screening methods are also provided.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/487,933 filed Jun. 4, 2012 which claims priorityfrom U.S. Provisional Application Ser. No. 61/492,680 filed Jun. 2,2011, entitled “AMYOTROPHIC LATERAL SCLEROSIS DETECTION DEVICES,METHODS, AND BIOMARKERS”, the complete disclosure of which said twoapplications is hereby incorporated by reference in its entirety.

GOVERNMENT SUPPORT

The invention described herein was made with government support undergrant number DGE0549500 awarded by the National Science Foundation(NSF). Accordingly, the United States has certain rights in theinvention.

FIELD OF THE INVENTION

The invention provides a quantum dot (QD) modified optical fiber-basedbiosensor which characterizes matrix metalloproteinase (MMP) enzymeactivity at pain signaling sites in the central nervous system (CNS) invivo. Related systems and peptide biomarker screening methods are alsoprovided.

BACKGROUND OF THE INVENTION

Given that existing pain treatments (which primarily target neurons)reduce pain by only around 25-40% in less than half of the 15 millionpatients suffering from chronic neuropathic pain in the US, there is aneed for new methods to identify and investigate pain-related cellularprocesses beyond only neuronal function.

Of the pain drugs currently available, opioid analgesics are the goldstandard despite their addiction liabilities. A biomarker forneuropathic pain does not exist despite the need to objectively identifythose individuals in need of treatment.

Further, the neuron centered view of pain processing is changing, andnon-neuronal targets are emerging, including glial cells and leukocytesthat enrich the spinal cord and other central nervous system sites (CNS)critical for pathological pain signaling. Thus, it is possible that bytargeting non-neuronal signaling mechanisms, a novel biomarker toidentify neuropathic pain may emerge.

Indeed, upon strong pain-related neuronal activation, spinal cord gliacontribute to persistent pathological pain by responding to andreleasing proinflammatory cytokines like IL-1β & TNF-α as well as theactivity of matrix metalloproteinase (MMP) enzymes [Dev 2010].Leukocytes may additionally contribute to ongoing pathological pain byreleasing and responding to IL-1β and TNF-α, with an additionalcontribution of MMPs. However, MMPs not only contribute to theneuroinflammation, but may be directly involved in pain-associated nervedamage.

There has been an increasing interest in matrix metalloproteinase (MMP)enzyme biosensing activity as a biomarker for neurological diseases.Several studies on MMP activity in patients with neurological diseasesgenerally indicate abnormal MMP activity in the serum and CSF. Inspecific, patients with amyotrophic lateral sclerosis (ALS)statistically show 3 times lower MMP-9 activity in the CSF and 2 timeshigher MMP-9 activity in the serum, in comparison to healthy controls[Niebroj-Dobosz 2010]. Furthermore, it was statistically shown thatpatients with HIV [Liuzzi 2000] and multiple sclerosis [Leppert 1998]have increased MMP-9 activity, while patients with Alzheimer's show noabnormal MMP-9 activity at all [Adair 2004]. Most of these findings weredone via gel electrophoresis; a multistep assay process. A simpler andhighly effective sensing method for indicating these findings could havea positive impact on clinical treatment.

MMPs are zinc dependent endopeptidases which can bind to specificnon-terminal amino acids in peptides with specific amino acid sequences.Active MMPs form bonds to amino acid residues at the active site of theMMP, which consists of a highly reactive zinc ion. After binding to apeptide, MMPs will catalyze hydrolysis and break a bond between twoamino acid residues in a peptide. MMPs are initially synthesized asinactive by the body's immune system, but become active due to injury,inflammation, or the presence of other proteinases, foreign bodies, andpathogens. Specifically, MMPS are made active by the removal of apeptide blocking the active region due to a bond between the Zn²⁺ ionand the thiol group in the cysteine amino acid residue.

The previous findings reported in literature relating neurologicaldiseases to MMPs are a good indication of their potential as abiomarker. However, in vivo biosensing of MMP activity remains largelyunexplored.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a quantum dot (QD) modifiedoptical fiber-based biosensor system adapted for evaluation ofmetalloproteinase (MMP) enzyme activity at pain signaling sites in thecentral nervous system (CNS), the system comprising:

(a) a multimode silica optical fiber comprising one or more quantum dotsthat (1) are bioconjugated on their surface to one or more peptideshaving an affinity for the metalloproteinase (MMP) enzyme, and (2) thatare tethered to an insertion end of said multimode silica optical fiberby one or more silane coupling agents; and(b) a photon detector;wherein changes in photon emission resulting from interaction of themetalloproteinase (MMP) enzyme and peptides having an affinity for themetalloproteinase (MMP) enzyme are detected by the photon detector.

In a preferred embodiment, the metalloproteinase (MMP) enzyme activitydetected by the system described above is implicated in neuroimmuneneuropathies. In certain embodiments, neuroimmune neuropathies includeamyotrophic lateral sclerosis (ALS) and multiple sclerosis.

In another embodiment, the insertion end of the multimode silica opticalfiber is adapted for insertion into an in-dwelling spinal catheter thatis submerged into either the epidermal or intrathecal regions of thecentral nervous system.

Preferably, in systems and methods of the invention:

(1) the quantum dots do not contain cadmium;

(2) changes in photon emission resulting from interaction of saidmetalloproteinase (MMP) enzyme and peptides having an affinity for saidmetalloproteinase (MMP) enzyme are reflected by measurable perturbationsin quantum dot fluorescence and are detected by a transducer of saidphoton detector;(3) the silane coupling agents comprise a thiol functional group thatcovalently bonds to metallic surface; and(4) quantum dots are either colloidal Mn-doped ZnSe/ZnS quantum dots,Cu-doped ZnSe quantum dots or InP/ZnSe quantum dots.

In one embodiment of the systems and methods of the invention:

(a) the one or more peptides having an affinity for themetalloproteinase (MMP) enzyme are modified at either their N orC-terminals with a fluorescent protein or dye;

(b) in operation, the fluorescent protein or dye undergoes Försterresonant energy transfer (FRET) with the quantum dots, the Försterresonant energy transfer (FRET) being disrupted by interaction of saidmetalloproteinase (MMP) enzyme and peptides having an affinity for themetalloproteinase (MMP) enzyme; and(c) the disruption is (1) detected by said photon detector, and (2)correlated as an indicator of metalloproteinase (MMP) enzyme activity.

In a preferred embodiment, the photon detector is adapted for thedetection of ratiometric changes that occur as quantum dot fluorescenceincreases and protein fluorescence decreases due to metalloproteinase(MMP) enzyme activity, and in operation the detected ratiometric changesare correlated as an indicator of metalloproteinase (MMP) enzymeactivity in vivo.

In another embodiment, the invention provides a method for evaluating invivo metalloproteinase (MMP) enzyme activity at a subject's centralnervous system pain signaling site, the method comprising:

(a) providing a quantum dot (QD) modified optical fiber-based biosensorsystem as described herein;

(b) inserting the multimode silica optical fiber of the biosensor systeminto an in-dwelling spinal catheter that is submerged into either theepidermal or intrathecal regions of the subject's central nervoussystem; and

(c) measuring changes in photon emission resulting from interaction ofsaid metalloproteinase (MMP) enzyme and peptides having an affinity forsaid metalloproteinase (MMP) enzyme, said changes having been detectedby said photon detector.

Förster resonant energy transfer (FRET) disruption techniques asdescribed herein can also be used in the diagnostic methods of theinvention. Detection of ratiometric changes that occur as quantum dotfluorescence increases and protein fluorescence decreases due tometalloproteinase (MMP) enzyme activity as described herein provideanother aspect of the disclosed diagnostic methods.

In still another embodiment, the invention provides a method foridentifying a peptide having an affinity for a metalloproteinase (MMP)enzyme implicated in a neuroimmune neuropathy, the method comprising:

(a) providing a quantum dot (QD) modified optical fiber-based biosensorsystem of as described herein;

(b) inserting the multimode silica optical fiber of the biosensor systeminto an in-dwelling spinal catheter that is submerged into either theepidermal or intrathecal regions of a subject's central nervous system;and

(c) measuring any changes in photon emission that result frominteraction of said metalloproteinase (MMP) enzyme and said peptide, asdetected by said photon detector; and

(d) comparing any changes in photon emission to a control value.

These and other embodiments of the invention are described further inthe Detailed Description of the Invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an illustration depicting hydroxyl modified silica.

FIG. 2 is an illustration depicting mercaptopropyltrimethoxysilane(MPTMS) modified silica.

FIG. 3 is an illustration depicting a QD covalently bonded to silica bymeans of the MPTMS silane coupling agent.

FIG. 4 is an illustration of the optical setup for performing QDmodified optical fiber-based biosensing measurements for MMP activity,in vivo.

FIG. 5 is an illustration depicting a fluorescently excited QDcovalently bonded to silica with a bioconjugated MMP substrate peptideat the surface.

FIG. 6 is an illustration of a sensing method depicting the cleaved MMPsubstrate peptide resulting in altered QD fluorescence.

FIG. 7 is a normalized PL excitation spectrum and a raw PL excitationspectrum for atomic Mn transitions for hydrophobic Mn-doped ZnSe withZnS shell QDs in toluene.

FIG. 8 is a PL emission spectrum for atomic Mn transitions forhydrophobic Mn-doped ZnSe with ZnS shell QDs in toluene.

FIG. 9 is a normalized PL excitation spectrum and a raw PL excitationspectrum of ZnS defects for hydrophobic Mn-doped ZnSe with ZnS shell QDsin toluene.

FIG. 10 is a PL emission spectrum of ZnS defects for hydrophobicMn-doped ZnSe with ZnS shell QDs in toluene.

FIG. 11 is a normalized PL excitation spectrum for atomic Mn transitionsfor hydrophilic Mn-doped ZnSe with ZnS shell QDs in water.

FIG. 12 is a PL emission spectrum for atomic Mn transitions forhydrophilic Mn-doped ZnSe with ZnS shell QDs in water.

FIG. 13 is an illustration depicting Förster resonance energy transfer(FRET) between a QD donor tethered to silica and a fluorescent protein,or dye, acceptor.

FIG. 14 is an illustration depicting FRET disruption between a QD donortethered to silica and a fluorescent protein, or dye, acceptor.

FIG. 15 is an illustration depicting FRET between a tethered QD donorand a tethered QD acceptor.

FIG. 16 is an illustration depicting FRET disruption between a tetheredQD donor and a tethered QD acceptor.

FIG. 17 is a PL spectrum depicting overlap between the emission spectrumof the Cu-doped ZnSe QD donor and the excitation spectrum of theCu-doped InP/ZnSe QD acceptor.

DETAILED DESCRIPTION OF THE INVENTION

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” include plural referents unlessexpressly and unequivocally limited to one referent. Thus, for example,reference to “a compound” includes two or more different compound. Asused herein, the term “include” and its grammatical variants areintended to be non-limiting, such that recitation of items in a list isnot to the exclusion of other like items that can be substituted orother items that can be added to the listed items.

As described in U.S. Pat. No. 8,170,665, “quantum dots (QDs) aresemiconductor nanoparticles that were discovered in the early 1980's.Certain known QDs used for biological applications consist of a cadmiumselenide or cadmium tellurium semiconductor core, a zinc sulfide innershell and an outer polymer coating. The result is a water-solubleparticle 13-15 nm in diameter.

Similar to organic fluorophores, QDs absorb photons of light of onewavelength and emit light of a different wavelength. Traditionalfluorophores use absorbed energy to transfer electrons to excited statesand energy is released in the form of fluorescent light when theseelectrons return to their resting states. When electrons move todifferent energy levels in QDs, they behave analogously, generatingelectron holes called excitons. The quantum system of excitons makes QDfluorescence much brighter and more photostable (less prone tophotobleaching) than traditional fluorophores.

The energy state of an exciton dictates the wavelength of light emittedby a particular QD after excitation. QDs have a unique property known astunability, wherein the physical size of the QD determines thewavelength of emitted light. Smaller dots emit blue fluorescent lightand as the core size of the dots increases, emitted light becomesredder. Another important feature that distinguishes QDs fromconventional fluorescent dyes is the large distance between thewavelength of excitation and emission light. This energy difference,known as the Stokes' shift, means that QDs can be excited by ultravioletlight at a wavelength much lower than the peak emission wavelength.Thus, QDs can be excited by any wavelength lower than its emissionwavelength. Therefore, particles are excited and emitted light iscollected in a very efficient manner.”

Preferred quantum dots used in the instant invention include but are notlimited to Mn-doped ZnSe/ZnS quantum dots, Cu-doped ZnSe quantum dotsInP/ZnSe quantum dots and colloidal Mn-doped ZnSe/ZnS quantum dots.These quantum dots ideally emit light at a wavelength of between about498 nm to about 750 nm.

Bioconjugated QD probes, which are linked to biological molecules likemonoclonal antibodies, peptides, proteins, or nucleic acids and containbright and stable fluorescent light emission and multiplexing potential(i.e., capability to detect multiple disease markers simultaneously),provide a novel highly sensitive approach to detect low-abundant copynumbers of potential disease biomarkers (e.g., nucleic acids andproteins) in bodily fluid and tissue samples. QDs with their intrinsichigh spatial resolution and sensitivity of fluorescence imaging can notonly serve as sensitive probes for disease biomarkers, but they couldalso enable the detection of hundreds to thousands of simultaneously(i.e. multiplexing). See U.S. Patent Application Document No.20070157325. Those of ordinary skill in the art know how to bioconjugatepeptides having an affinity for said metalloproteinase (MMP) enzyme to aquantum dot.

A “multimode silica optical fiber” can include a wide-variety of silicaoptical fibers known as being useful in vivo diagnostic applications,e.g. around 100 to around 400 μm core diameter (as far as I understand,larger core diameters are needed in order to transmit visible lightthrough silica multi-mode fibers, which are typically 50 μm and designedfor ˜850-1350 nm light, where I would need visible light according tothe characteristics of the QDs presented in this patent) fused silicaoptical fibers.

“Silane coupling agents” include but are not limited totetraethoxysilane (TEOS), γ-mercaptopropyltrimethoxysilane (MPS),γ-mercaptopropyltriethoxysilane, γ-aminopropyltriethoxysilane (APS),3-thiocyanatopropyltriethoxysilane, 3-glycidyloxypropyltriethoxysilane,3-isocyanatopropyltriethoxysilane, and3-[2-(2-aminoethylamino)ethylamino]propyl-triethoxysilane.

The order of magnitude of the affinity between a peptide andmetalloproteinase (MMP) enzyme can vary between approximately 10⁻¹⁰ M toaround 10⁻⁵ M, and preferentially is between approximately 0.1 nM andapproximately 100 nM. The term approximately is used to cover thevariability in the measurement of the affinity, with variation typicallybeing from around 5% to 10% measurement error.

“Photon detectors include but are not limited to photodetectors, lightdetectors, photon counters. In a non-limiting example, photon detectorscan comprises at least one three-dimensional (3D) photonic crystal thatoperates at a relatively low temperature and is formed or grown on aglass substrate, and at least one field emission transistor (FET), e.g.,a thin-film transistor (TFT), that is also formed on a glass substrate(e.g., a TFT can have a thin film of silicon, and the transistors arefabricated using this thin layer). In at least one further exemplaryembodiment, suitable types of transistors other than FETs can beemployed. In at least one exemplary embodiment, the PDC is also referredto as a detection layer. In at least one exemplary embodiment, thephoton detector is a digital photon detector. In at least one otherexemplary embodiment of the invention, glass is the substrate, althoughany other compatible substrates providing the optical propertiesnecessary for the present invention (e.g., transparent plastics) arealso contemplated.

Scintillation crystals coupled to avalanche photo diodes can also beused as photon detectors. In other embodiments, scintillation crystalsare coupled with photomultiplier tubes. The scintillation crystals arebismuth germanium oxide, gadolinium oxyorthosilicate, or lutetiumoxyorthosilicate crystals, but other crystals may be used.

Photon detectors can be arranged individually or in groups. Thedetectors can generate analog signals, position signals or energysignals. Each of the signals can output as a differential signal pair.

Neuroimmune activation have been shown to play a role in the etiology ofa variety of neurological disorders such as stroke, Parkinson's andAlzheimer's disease, multiple sclerosis, pain, and AIDS-associateddementia. However, cytokines and chemokines also modulate CNS functionin the absence of overt immunological, physiological, or psychologicalchallenges. Essentially any cytokines and cytokine receptor-relateddisorder is susceptible to diagnosis and screening using the systems andmethods of the invention. Chronic pain is an extremely debilitatingdisease syndrome for which current treatment modalities are largelyineffective. Neuroimmune activation in the maintenance of chronic painis another process that may be analyzed using the systems and methods ofthe invention. Further, disorders implicating a pathway that linksperipheral neuronal injury/inflammation with the activation of centralnervous system neuroglial cells, which contributes to sustained neuronalhyper-excitability, are also subject to assessment by the systems andmethods described herein. Preferred systems and methods diagnose andscreen for peptide markers for amyotrophic lateral sclerosis (ALS) andmultiple sclerosis.

The MMP family of zinc endopeptidases (24 individual enzymes in humans)includes collagenases, gelatinases, matrilysins, stromelysins andmembrane-type MMPs. MMP proteolysis regulates the levels and thefunctionality of extracellular matrix components and cell surfacesignaling receptors. In the damaged nerves, MMP proteolysis can be bothdetrimental and beneficial to axonal growth and recovery of neuronalfunction. In peripheral adult nerves, MMP-9 (gelatinase B) is producedonly after injury. After a lesion, MMP-9 is produced by myelinating SCs(mSCs), immune and endothelial cells to promote the breakdown of themyelin sheath, the blood-nerve barrier and the SC basal lamina. MMP-9 isa multi-domain enzyme with wide-ranging substrate preferences. Earlierwork suggests that MMP-9 controls the phenotypic switching in SCs byactivation of the extracellular-signal-regulated kinase (ERK)1/2 via theneuregulin/ErbB and insulin growth factor (IGF)-1 ligand/receptorsystems. As a result MMP-9 suppresses 5-bromo-2-deoxyuridine (BrdU)incorporation in cultured primary SC and the injured nerves.

MMP's such as MMP-9 are relatively well-characterized (see e.g. MMP-9UNIPROT listing) and those of ordinary skill in the art can employwell-known techniques to identify “peptides having an affinity for saidmetalloproteinase (MMP) enzyme”. The paper group from the paper by Turkhave identified 6 different MMP peptides. See. e.g. Xu, et al.,http://www.biochemj.org/bj/imps_x/pdf/BJ20050650.pdf. The followingpeptide ligands are from [Turk 2001], however the ‘C’ is the cysteineresidue (thiol containing molecule) used to link the peptide to the QDsurface. Also, the ‘X’ in MTI-MMP means that there is no preferredselectivity for that position in the peptide. The three dashes (---) isthe bond that is cleaved (the scissile bond). The six peptides arepresented hereinbelow. They may be used in various aspects of theinvention either alone or in combination. Other peptides, as describedabove, may also be used in the present invention, including thosepeptides which are identified pursuant to certain methods as describedherein.

MMP-7 substrate (matrilysin): (SEQ ID NO: 1) P-V-P-L-S---L-V-M-(C) PVPLSLVMC  MMP-1 substrate (Collagenase-1): (SEQ ID NO: 2)V-V-P-M-S---M-M-A-(C) VVPMSMMAC  MMP-2 substrate (Gelatinase A):(SEQ ID NO: 3) D-I-P-V-S---L-R-S-(C) DIPVSLRSC MMP-9 substrate (Gelatinase B): (SEQ ID No: 4) V-V-P-L-S---L-R-S-(C)VVPLSLRSC  MMP-3 substrate (Stromelysin-1): (SEQ ID NO: 5)N-K-P-F-S---M-M-M-(C) NKPFSMMMC  MTI-MMP substrate (MMP-14):(SEQ ID NO: 6) F-I-P-X-S---L-R-M-(C) FIPXSLRMC 

“Evaluation of metalloproteinase (MMP) enzyme activity at pain signalingsites in the central nervous system (CNS) in real time” can include anassessment over a period of around 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1days, or around 48-36, or around 36-24, or around 24-12, or around 12-6,or around 6-1 hours, or around 60-45, or 45-30, or 30-15, or less than15 minutes after a subject presents for diagnosis or begins treatment.

As used herein, the terms “peptide” or “polypeptide” refers broadly to apolymer of two or more amino acids joined together by peptide bonds. Theterm “polypeptide” also includes molecules which contain more than onepolypeptide joined by a disulfide bond, or complexes of polypeptidesthat are joined together, covalently or noncovalently, as multimers (eg., dimers, tetramers). Thus, the terms peptide, oligopeptide, andprotein are all included within the definition of polypeptide and theseterms are used interchangeably. It should be understood that these termsdo not connote a specific length of a polymer of amino acids, nor arethey intended to imply or distinguish whether the polypeptide isproduced using recombinant techniques, chemical or enzymatic synthesis,or is naturally occurring.

Peptide and polypeptide sequences used in the invention also can begenerated by phage display. A randomnized peptide or protein can beexpressed on the surface of a phagemid particle as a fusion with a phagecoat protein. Techniques of monovalent phage display are widelyavailable (see, e.g., Lowman H. B. et al. (1991) Biochemistry30:10832-8.) Phage expressing randomized peptide or protein librariescan be panned with a solid matrix to which a AAAA molecule has beenattached. Remaining phage do not bind AAAA, or bind AAAA withsubstantially reduced affinity. The phage are then panned against asolid matrix to which a FRIP-1 has been attached. Bound phages areisolated and separated from the solid matrix by either a change insolution conditions or, for a suitably designed construct, byproteolytic cleavage of a linker region connecting the phage coatprotein with the randomized peptide or protein library. The isolatedphage can be sequenced to determine the identity of the selectedantagonist.

Where necessary, standard techniques for growing cells, separatingcells, and where relevant, cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques and protein syntheses are those known and commonlyemployed by those skilled in the art. A number of standard techniquesare described in Sambrook et al., 1989 Molecular Cloning, SecondEdition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis etal., 1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview,N.Y.; Wu (Ed.) 1993 Meth. Enzymol. 218, Part I; Wu (Ed.) 1979 Meth.Enzymol. 68; Wu et al., (Eds.) 1983 Meth. Enzymol. 100 and 101; Grossmanand Moldave (Eds.) 1980 Meth. Enzymol. 65; Miller (ed.) 1972 Experimentsin Molecular Genetics, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.; Old and Primrose, 1981 Principles of Gene Manipulation,University of California Press, Berkeley; Schleif and Wensink, 1982Practical Methods in Molecular Biology; Glover (Ed.) 1985 DNA CloningVol. I and II, IRL Press, Oxford, UK; Hames and Higgins (Eds.) 1985Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow andHollaender 1979 Genetic Engineering: Principles and Methods, Vols. 1-4,Plenum Press, New York. Abbreviations and nomenclature, where employed,are deemed standard in the field and commonly used in professionaljournals such as those cited herein.

High-content imaging techniques and diagnostic methods described hereincan use fluorescence-inducing compounds, e.g. a fluorescent moiety suchas a fluorescein dye or a rhodamine dye. In some embodiments, thefluorescent moiety comprises two or more fluorescent dyes that can actcooperatively with one another, for example by fluorescence resonanceenergy transfer (“FRET”). The fluorescent moiety may be any fluorophorethat is capable of producing a detectable fluorescence signal in anassay medium; the fluorescence signal can be “self-quenched” and capableof fluorescing in an aqueous medium. “Quench” refers to a reduction inthe fluorescence intensity of a fluorescent group as measured at aspecified wavelength, regardless of the mechanism by which the reductionis achieved. As specific examples, the quenching may be due to molecularcollision, energy transfer such as FRET, a change in the fluorescencespectrum (color) of the fluorescent group or any other mechanism. Theamount of the reduction is not critical and may vary over a broad range.The only requirement is that the reduction be measurable by thedetection system being used. Thus, a fluorescence signal is “quenched”if its intensity at a specified wavelength is reduced by any measurableamount.

Examples of fluorophores include xanthenes such as fluoresceins,rhodamines and rhodols, cyanines, phtalocyanines, squairanines, bodipydyes, pyrene, anthracene, naphthalene, acridine, stilbene, indole orbenzindole, oxazole or benzoxazole, thiazole or benzothiazole,carbocyanine, carbostyryl, prophyrin, salicylate, anthranilate, azulene,perylene, pyridine, quinoline, borapolyazaindacene, xanthene, oxazine orbenzoxazine, carbazine, phenalenone, coumarin, benzofuran, orbenzphenalenone. Examples of rhodamine dyes include, but are not limitedto, rhodamine B, 5-carboxyrhodamine, rhodamine X (ROX),4,7-dichlororhodamine X (dROX), rhodamine 6G (R6G),4,7-dichlororhodamine 6G, rhodamine 110 (R110), 4,7-dichlororhodamine110 (dR110), tetramethyl rhodamine (TAMRA) and4,7-dichlorotetramethylrhodamine (dTAMRA). Examples of fluorescein dyesinclude, but are not limited to, 4,7-dichlorofluoresceins,5-carboxyfluorescein (5-FAM) and 6-carboxyfluorescein (6-FAM).

The practice of the present invention may also employ conventionalbiology methods, software and systems. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes and etc. The computer executable instructions may bewritten in a suitable computer language or combination of severallanguages. Basic computational biology methods are described in, forexample Setubal and Meidanis et al., Introduction to ComputationalBiology Methods (PWS Publishing Company, Boston, 1997); Salzberg,Searles, Kasif, (Ed.), Computational Methods in Molecular Biology,(Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics:Application in Biological Science and Medicine (CRC Press, London, 2000)and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysisof Gene and Proteins (Wiley & Sons, Inc., 2.sup.nd ed., 2001). See U.S.Pat. No. 6,420,108.

The present invention may also make use of various computer programproducts and software for a variety of purposes, such as probe design,management of data, analysis, and instrument operation. See, U.S. Pat.Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555,6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.

Additionally, the present invention relates to embodiments that includemethods for providing information over networks such as the Internet.For example, the components of the system may be interconnected via anysuitable means including over a network, e.g. the ELISA plate reader tothe processor or computing device. The processor may take the form of aportable processing device that may be carried by an individual usere.g. lap top, and data can be transmitted to or received from anydevice, such as for example, server, laptop, desktop, PDA, cell phonecapable of receiving data, BLACKBERRY™, and the like. In someembodiments of the invention, the system and the processor may beintegrated into a single unit. In another example, a wireless device canbe used to receive information and forward it to another processor overa telecommunications network, for example, a text or multi-mediamessage.

The functions of the processor need not be carried out on a singleprocessing device. They may, instead be distributed among a plurality ofprocessors, which may be interconnected over a network. Further, theinformation can be encoded using encryption methods, e.g. SSL, prior totransmitting over a network or remote user. The information required fordecoding the captured encoded images taken from test objects may bestored in databases that are accessible to various users over the sameor a different network.

In some embodiments, the data is saved to a data storage device and canbe accessed through a web site. Authorized users can log onto the website, upload scanned images, and immediately receive results on theirbrowser. Results can also be stored in a database for future reviews.

In some embodiments, a web-based service may be implemented usingstandards for interface and data representation, such as SOAP and XML,to enable third parties to connect their information services andsoftware to the data. This approach would enable seamless datarequest/response flow among diverse platforms and software applications.

In certain non-limiting embodiments, an increase or a decrease in asubject or test sample of the level of photon emission resulting frominteraction of said metalloproteinase (MMP) enzyme and peptides havingan affinity for said metalloproteinase (MMP) enzyme as compared to acomparable level of photon emission resulting from interaction of saidmetalloproteinase (MMP) enzyme and peptides having an affinity for saidmetalloproteinase (MMP) enzyme in a control subject or sample can be anincrease or decrease in the magnitude of approximately ±5,000-10,000%,or approximately ±2,500-5,000%, or approximately ±1,000-2,500%, orapproximately ±500-1,000%, or approximately ±250-500%, or approximately±100-250%, or approximately ±50-100%, or approximately ±25-50%, orapproximately ±10-25%, or approximately ±10-20%, or approximately±10-15%, or approximately ±5-10%, or approximately ±1-5%, orapproximately ±0.5-1%, or approximately ±0.1-0.5%, or approximately±0.01-0.1%, or approximately ±0.001-0.01%, or approximately±0.0001-0.001%.

The values obtained from controls are reference values representing aknown health status and the values obtained from test samples orsubjects are reference values representing a known disease status. Theterm “control”, as used herein, can mean a sample of preferably the samesource (e.g. blood, serum, tissue etc.) which is obtained from at leastone healthy subject to be compared to the sample to be analyzed. Inorder to receive comparable results the control as well as the sampleshould be obtained, handled and treated in the same way. In certainexamples, the number of healthy individuals used to obtain a controlvalue may be at least one, preferably at least two, more preferably atleast five, most preferably at least ten, in particular at least twenty.However, the values may also be obtained from at least one hundred, onethousand or ten thousand individuals.

Thus, in certain embodiments, the systems and methods of the inventionutilize QD-based biosensing methods to detect spinal MMP activity inpain-signaling sites during peripheral neuropathy, discreetly identifyMMP spinal cord enrichment at neuropathic pain-processing sites,identify and aid in the understanding of chronic neuroimmuneneuropathies, can develop time resolved analyte activity calibrationcurves, provide a means for real time in vivo biosensing withoutreleasing QDs into the biosensing environment, provide an in vivo methodto monitor protease activity by inserting the QD biosensing probethrough an in-dwelling catheter, monitor disease development andtreatment evaluation, provide QD-based biosensing device with QDsconsisting of duel fluorescent emissions that can be used in conjunctionwith one another to quantify and identify the analyte, can utilize themeasurable change between two different fluorescent emissions occurringwithin the same QD upon excitation as a way to quantify analyteactivity, can detect MMP activity from changes in QD fluorescence as aresult of MMPs clipping peptides bioconjugated to QDs coupled to asubstrate, and can quantify analyte activity from the measurable changein fluorescence due to FRET disruption between a QD donor and a QDacceptor as a result of analyte detection.

These and other aspects of the invention are illustrated further by thefollowing non-limiting examples.

Example 1 Quantum Dot (QD) Modified Optical Fiber-Based Biosensor System

QDs are immobilized onto the silica surface of an optical fiber tip, viathe use of the silane coupling agent, 3-mercaptopropyltrimethoxysilane(MPTMS), which contains a thiol functional group that covalently bondsto metallic surfaces, due to a known affinity between thiol moleculesand metal. Hydrolyzable regions of MPTMS react with hydroxyl groups andform stable oxane bonds, and so a monolayer of the MPTMS silane couplingagent can be formed over hydroxyl modified silica [Hu 2001]. In FIG. 1,silica 1 is modified with hydroxyl groups 2, and in FIG. 2 MPTMS reactswith hydroxyl groups to form an MPTMS monolayer 3 with thiol moleculesat the surface. In FIG. 3, Due to the aforementioned metal affinity fromthe thiol molecules, QDs 4 will be immobilized and covalently bonded tothe thiol monolayer from the MPTMS. The optical setup in FIG. 4 for thebiosensor will consist of a fiber optic laser setup with a PMT detector11 and a Y-fiber (fiber coupler) 8 for duel entrance and exit of theexcitation 9 and emission 10 lights, respectively. A fiber connector 7is used to connect the QD tip-modified optical fiber 6 to the opticalsetup. The QD modified fiber is inserted through an in-dwelling catheter5 in order to make MMP enzyme biosensing measurements in the intendedmedium.

Due to the highly sensitive surface properties of QDs, the opticalproperties of the photoluminescence can change upon surfacemodification. Changes in fluorescence emission can result when moleculesare conjugated to the surface of QDs and further changes in fluorescencecan occur when these conjugated molecules are modified. Similarly,bioconjugating peptides to the surface of QDs can affect the florescenceemission of QDs, such as a change in the wavelength, and with MMPenzymes cleaving the bioconjugated peptides, an additional change influorescence can occur. It is the change in QD fluorescence propertiesthat can occur from the MMPs cleaving peptides that is intended to beused and interpreted as a quantifiable analysis of MMP activity.

FIG. 5 and FIG. 6 illustrate changes in fluorescence as a result ofactive MMPs cleaving peptides. In FIG. 5, a QD 4 tethered to silica 1from the MPTMS coupling agent 3 with the MMP peptide substrate 12conjugated to its surface emits fluorescence 14 when excited by light 13at a particular wavelength. In FIG. 6, the QD 15, illuminated by thesame excitation light wavelength, undergoes a change in its opticalproperties and fluorescently emits light 17 at a different wavelengthafter the peptide 16 has been cleaved from an active MMP enzyme.

The effects that peptide conjugation has on the optical properties ofdoped non-cadmium containing QDs will be utilized as a means for MMPbiosensing. Preliminary experiments involving synthesis and opticalmeasurements of Mn-doped ZnSe/ZnS colloidal QDs have revealed a notablechange in the photoluminescence properties between the synthesized QDsbefore and after applying a hydrophilic ZnS shell synthesis, suggestingsurface sensitive effects to the QD emission.

Mn-doped ZnSe/ZnS QDs were colloidally synthesized through modificationsof a synthesis protocol by [Pradhan 2005] and [Acharya 2010], which wereoptically characterized with a spectrofluorometer. It was subsequentlylearned that the organically synthesized QDs exhibited 2photoluminescence peaks, which emitted simultaneously two differentemissions at a single excitation wavelength, with one emission being dueto the dopant and the other being attributed to ZnS shell defects. FIG.7 and FIG. 8 are the excitation and emission spectra for one of thetransitions that take place, respectively, for Mn-doped ZnSe QDs intoluene. By using an excitation wavelength of 418 nm, two emissionfeatures show at 587 nm and 496 nm wavelengths. The 587 nm emission isbelieved to be from an atomic energy transition that takes place with Mndopant and the 496 nm emission being from ZnS defects. By using 453 nmexcitation wavelength the ZnS defect emission can be isolated. FIG. 9and FIG. 10 are the excitation and emission spectra, respectively, forthe ZnS defect transition that takes place for the Mn-doped ZnSe/ZnSQDs. Only the defect emission at the 497 nm wavelength, from the 453 nmexcitation wavelength, can be observed in FIG. 10.

Upon applying a hydrophilic ZnS shell synthesis, based off of amodification by [Li 2008] to the Mn-doped ZnSe QDs, in which QDs couldbe made dispersable in water due to charged carboxyl groups at thesurface, the 497 nm emission line would no longer show, suggestingsurface sensitivity to this particular transition. Consequently, Thehydrophillic QDs would only exhibit an emission peak at 590 nm, and witha significant blue shift of the excitation wavelength. FIG. 11 and FIG.12 show the excitation and emission spectra of the hydrophillic QDs,respectively, where only the 597 nm Mn emission can be observed. Thehydrophilization involves a thiol containing molecule that attaches tothe surface, and the result is that the 497 nm ZnS defect emissionvanishes. Therefore, by attaching peptides to the QDs from a thiol basedbond, the 497 ZnS defect emission will subsequently disappear, orweaken, and will be progressively recovered as a result of MMPs cleavingthe peptides. The ratio of orange 587 nm emission to that of recovered497 nm emission will be the basis for the optical transducer mechanismfor measuring MMP activity with QDs.

Example 2 Förster Resonant Energy Transfer (FRET)

QD-modified optical fiber based MMP biosensing in accordance with theinvention may also emply Förster resonant energy transfer (FRET) betweenQD donors and fluorescent peptide, or dye, acceptors. The embodimentconsists of QDs covalently linked to silica optical fiber tip surfaceswith MMP substrate peptides, modified at either the N or C-terminalswith fluorescent proteins or dyes, covalently linked to the QDs. Afluorescent protein with an absorption spectrum that overlaps theemission spectrum of the QD will result in FRET, when the 2 fluorophoresare in close enough vicinity. This means that the protein will fluorescedue to an energy transfer that occurs from the QD emission while the 2fluorophores are being illuminated with light at a wavelength thatexcites the QD only. In FIG. 13, light 13 that excites the QD 4 resultsin an energy transfer (FRET) 18 to a fluorescent protein, or dye, 20that is linked to the QD surface, via the MMP peptide substrate 12, andfluorescence emission 21 from the protein results. As a sensing method,it is intended to utilize the FRET disruption that results from thecleaving of the peptide from the targeted MMP enzyme as a measurableparameter for enzyme activity. In FIG. 14, the peptide 16 is cleavedfrom the MMP enzyme, the fluorescent protein 20 falls out of range forFRET to occur and the fluorescence from the protein, or dye, 20diminishes and is replaced by the QD fluorescence 14. In an ensemble ofQDs with conjugated fluorescence proteins, the ratiometric change thatoccurs as QD fluorescence increases and protein fluorescence decreases,due to MMP activity, can be used as a reliable quantifiable method ofmeasuring MMP activity under in vivo conditions.

Example 3 QD FRET Acceptors and Donors Attached with a Single Peptide

A QD modified optical fiber-based MMP biosensor of the invention canalso employ a QD FRET acceptors and donors attached with a singlepeptide, without the use of fluorescent peptides or dyes. The opticalfiber modification involves tethering QD Forster Resonance EnergyTransfer (FRET) pairs linked together with a peptide onto the tip of amultimode silica optical fiber, via a silane coupling agent. In FIG. 15,a peptide sequenced for the particular MMP of interest 12 is used tolink a QD donor 13 and a QD acceptor 25, that are both tethered tosilica 1, by means of the affinity interaction between biotin 23 and astreptavidin 22 modified silane coupling agent 21. The short distancebetween the QD donor and acceptor as a result of the peptide linkerresults in FRET 18 between the 2 QD fluorophores. The excitation light13 transfers energy from the QD donor 24 to the QD acceptor 25 resultingin the fluorescence 26 of the acceptor. In FIG. 16, Quantitativeratio-metric data involving MMP activity can be recorded based on theFRET disruption, which results from active MMPs in the CSF clipping thepeptide 16 molecules linking the illuminated QD pairs. The FRETdisruption causes fluorescence 27 in the QD donor 24 and a decrease orelimination to the fluorescence in the QD acceptor 25. The degree ofchange between the intensity of the donor emission to that of theacceptor is used to characterize MMP activity.

Thermally excited electrons and lattice vibrations that result fromheated QDs contribute to a significant loss in PL intensity and quantumefficiency. The intended application involves biosensing inside of thehuman body (˜37° C.) from QD FRET pairs and the loss of PL intensity atthe elevated body temperature is perceived as a potential problem. Inorder to maintain better thermal PL stability a synthesis by [Acharya2010] and [Pradhan 2005] for green emitting doped ZnSe QD donors and asynthesis by [Xie 2009] for red emitting doped InP QD acceptors wasmodified in order to achieve QD luminescence with little loss at theelevated body temperature. The low PL loss may be due to the Cu impuritythat is doped into the nanocrystal lattice which is due to atomictransitions that aren't as coupled to the lattice vibrations [Xie 2009].

The PL spectral data for Cu-doped ZnSe and InP/ZnSe QDs have sufficientspectral overlap for FRET to occur between the donor and acceptor QDs.In FIG. 17, the emission spectrum 29 of the Cu-doped ZnSe donor overlapsthe excitation spectrum 30 of the Cu-doped InP QD acceptor. Theexcitation spectrum of the donor 28 overlaps the excitation spectrum 30of the acceptor, and the emission spectrum of the donor 29 and theemission spectrum of the acceptor 31 are well separated and theratiometric change between the emission intensities of the donor andacceptor is the parameter used to quantify MMP activity, in vivo.

REFERENCES

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What is claimed is:
 1. A quantum dot (QD) modified optical fiber-basedbiosensor system adapted for evaluation of metalloproteinase (MMP)enzyme activity at pain signaling sites in the central nervous system(CNS), said biosensor system comprising: a multimode silica opticalfiber comprising one or more quantum dots that are bioconjugated ontheir surface to one or more peptides having an affinity for saidmetalloproteinase (MMP) enzyme, and that are tethered to an insertionend of said multimode silica optical fiber by one or more silanecoupling agents; and a photon detector; wherein changes in photonemission resulting from interaction of said metalloproteinase (MMP)enzyme and peptides having an affinity for said metalloproteinase (MMP)enzyme are detected by said photon detector, said multimode silicaoptical fiber is tethered to a quantum dot donor and to a quantum dotacceptor by one or more biotin and streptavidin-modified silane couplingagents; and said quantum dot donor and quantum dot acceptor are linkedby a peptide having an affinity for said metalloproteinase (MMP) enzyme.2. A method for evaluating in vivo metalloproteinase (MMP) enzymeactivity at a subject's central nervous system pain signaling site, themethod comprising: a) providing a quantum dot (QD) modified opticalfiber-based biosensor system adapted for evaluation of metalloproteinase(MMP) enzyme activity at pain signaling sites in the central nervoussystem (CNS), said biosensor system comprising: a multimode silicaoptical fiber comprising one or more quantum dots that (1) arebioconjugated on their surface to one or more peptides having anaffinity for said metalloproteinase (MMP) enzyme, and (2) that aretethered to an insertion end of said multimode silica optical fiber byone or more silane coupling agents; and a photon detector; whereinchanges in photon emission resulting from interaction of saidmetalloproteinase (MMP) enzyme and peptides having an affinity for saidmetalloproteinase (MMP) enzyme are detected by said photon detector, theone or more peptides having an affinity for said metalloproteinase (MMP)enzyme are modified at either their N or C-terminals with a fluorescentprotein or dye, in operation, the fluorescent protein or dye undergoesFörster resonant energy transfer (FRET) with the quantum dots, saidFörster resonant energy transfer (FRET) being disrupted by interactionof said metalloproteinase (MMP) enzyme and peptides having an affinityfor said metalloproteinase (MMP) enzyme; and wherein said disruption isdetected by said photon detector, and correlated as an indicator ofmetalloproteinase (MMP) enzyme activity; b) inserting the multimodesilica optical fiber of the said biosensor system into an in-dwellingspinal catheter that is submerged into either the epidermal orintrathecal regions of the subject's central nervous system; and c)measuring changes in Förster resonant energy transfer (FRET) disruptioncaused by interaction of said metalloproteinase (MMP) enzyme andpeptides having an affinity for said metalloproteinase (MMP) enzyme,said changes having been detected by said photon detector.
 3. The methodaccording to claim 2 wherein the method is conducted in high-throughputformat and in real-time.
 4. A method for evaluating in vivometalloproteinase (MMP) enzyme activity at a subject's central nervoussystem pain signaling site, the method comprising: a) providing aquantum dot (QD) modified optical fiber-based biosensor system adaptedfor evaluation of metalloproteinase (MMP) enzyme activity at painsignaling sites in the central nervous system (CNS), said biosensorsystem comprising: a multimode silica optical fiber comprising one ormore quantum dots that (1) are bioconjugated on their surface to one ormore peptides having an affinity for said metalloproteinase (MMP)enzyme, and (2) that are tethered to an insertion end of said multimodesilica optical fiber by one or more silane coupling agents; and a photondetector; wherein changes in photon emission resulting from interactionof said metalloproteinase (MMP) enzyme and peptides having an affinityfor said metalloproteinase (MMP) enzyme are detected by said photondetector, the one or more peptides having an affinity for saidmetalloproteinase (MMP) enzyme are modified at either their N orC-terminals with a fluorescent protein or dye which undergoes Försterresonant energy transfer (FRET) with the quantum dots, said Försterresonant energy transfer (FRET) being disrupted by interaction of saidmetalloproteinase (MMP) enzyme and peptides having an affinity for saidmetalloproteinase (MMP) enzyme; wherein said disruption is detected bysaid photon detector, and correlated as an indicator ofmetalloproteinase (MMP) enzyme activity, the photon detector beingadapted for the detection of ratiometric changes that occur as quantumdot fluorescence increases and protein fluorescence decreases due tometalloproteinase (MMP) enzyme activity, and wherein in operation saiddetected ratiometric changes are correlated as an indicator ofmetalloproteinase (MMP) enzyme activity in vivo b) inserting themultimode silica optical fiber of the said biosensor system into anin-dwelling spinal catheter that is submerged into either the epidermalor intrathecal regions of the subject's central nervous system; and c)measuring ratiometric changes that occur as quantum dot fluorescenceincreases and protein fluorescence decreases due to metalloproteinase(MMP) enzyme activity, said changes having been detected by said photondetector.
 5. The method according to claim 4 wherein the method isconducted in high-throughput format and in real-time.
 6. A method foridentifying a peptide having an affinity for a metalloproteinase (MMP)enzyme implicated in a neuroimmune neuropathy, the method comprising: a)providing a quantum dot (QD) modified optical fiber-based biosensorsystem adapted for evaluation of metalloproteinase (MMP) enzyme activityat pain signaling sites in the central nervous system (CNS), saidbiosensor system comprising: a multimode silica optical fiber adaptedfor insertion into an in-dwelling spinal catheter that is submerged intoeither the epidermal or intrathecal regions of the central nervoussystem of a subject comprising one or more quantum dots that arebioconjugated on their surface to one or more peptides having anaffinity for said metalloproteinase (MMP) enzyme, and that are tetheredto an insertion end of said multimode silica optical fiber by one ormore silane coupling agents; and a photon detector; wherein changes inphoton emission resulting from interaction of said metalloproteinase(MMP) enzyme and peptides having an affinity for said metalloproteinase(MMP) enzyme are detected by said photon detector and the one or morepeptides having an affinity for said metalloproteinase (MMP) enzyme aremodified at either their N or C-terminals with a fluorescent protein ordye, in operation, the fluorescent protein or dye undergoes Försterresonant energy transfer (FRET) with the quantum dots, said Försterresonant energy transfer (FRET) being disrupted by interaction of saidmetalloproteinase (MMP) enzyme and peptides having an affinity for saidmetalloproteinase (MMP) enzyme, said disruption being (1) detected bysaid photon detector, and (2) correlated as an indicator ofmetalloproteinase (MMP) enzyme activity, wherein said photon detector isadapted for the detection of ratiometric changes that occur as quantumdot fluorescence increases and protein fluorescence decreases due tometalloproteinase (MMP) enzyme activity, and wherein in operation saiddetected ratiometric changes are correlated as an indicator ofmetalloproteinase (MMP) enzyme activity in vivo; b) inserting saidmultimode silica optical fiber of said biosensor system into saidin-dwelling spinal catheter; c) measuring any ratiometric changes thatoccur as a result of quantum dot fluorescence increases and proteinfluorescence decreases due to metalloproteinase (MMP) enzyme activity,as detected by said photon detector; and d) comparing any changes inphoton emission to a control value.
 7. The method according to claim 6wherein said silane coupling agents comprise a thiol functioning groupthat covalently bonds to a metallic surface.
 8. The method according toclaim 7 wherein said silane coupling agent is3-mercaptopropyltrimethoxysilane (MPTMS).
 9. The method according toclaim 6 wherein said biosensor contains a fiber optic laser comprising aPMT detector and a Y-fiber fiber coupler adapted for duel entrance andexit of excitation and emission lights, respectively and a fiberconnector is connected to the insertion end of said multimode silicaoptical fiber.
 10. The method according to claim 6 wherein the method isconducted in high-throughput format and in real-time.